1. Field of the Invention
The field of the invention relates to therapeutics. In particular, the field of the invention relates to the use of a redox-sensitive purine compound in combination with a redox agent to inhibit redox-sensitive GTPases, including Rho or Rab family GTPases.
2. Description of the Related Art
Small GTPases, including the Rho and Rab family GTPases, are involved in various cellular signaling events. Rho and Rab family GTPases belong to the Ras superfamily of GTPases. Rho and Rab GTPases, like all members of the Ras superfamily, function by cycling between inactive GDP-bound and active GTP-bound states (FIG. 1). Heo, J. (2011) Antioxid. Redox Signaling 14, 689-724. Various protein regulators such as guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins control this GDP/GTP cycling. GEFs enhance the guanine nucleotide exchange (GNE) of these GTPases. Bos et al. (2007) Cell 129, 865-877. Dbl's big sister (Dbs), one of the Rho-specific GEFs, has been shown to be a RhoC GEF. Dietrich et al. (2009) Biol. Chem. 390, 1063-1077. Because cells contain relatively higher concentrations of GTP than GDP (Traut, T. W. (1994) Mol. Cell. Biochem. 140, 1-22), the GEF-mediated GNE of these GTPases populates the GTP-loaded active GTPases in cells. GTPase-activating proteins stimulate hydrolysis of the γ-phosphate of the bound GTP to produce inactive GDP-bound GTPases and free phosphates. Bos et al. (2007) Cell 129, 865-877.
Several Rho family proteins have been identified thus far, including RhoA, RhoB, RhoC, RhoG, Rac1, Rac2, Rac3, Cdc42, TC10, TCL. Rho family members modulate various cellular processes, including cell morphology, movement and proliferation by mediating distinct cytoskeletal changes. Etienne-Manneville et al. (2002) Nature 420, 629-635. Misregulation of Rho GTPases has been implicated in many disorders, including cancer, heart and lung diseases, vascular diseases and diseases of the immune system. See e.g., van Leeuwen et al. (1995) Oncogene 11, 2215-2221; Fritz et al. (1999) Int. J. Cancer 81, 682-687; Boettner et al. (2002) Gene 286, 155-174; Benitah et al. (2003) Rev. Oncol. 5, 70-78; Numaguchi et al. (1999) Circ. Res. 85, 5-11; Laufs et al. (2000) Circ. Res. 87, 526-528; Satoh et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 7432-7437; Faried et al. (2006) Eur. J. Cancer. 42, 1455-1465; Touge et al. (2007) Int. J. Oncol. 30, 709-715.
RhoA has increasingly attracted clinical interest because of the emerging evidence of its role in the pathogenesis of several blood vessel diseases, including hypertension and atherosclerosis. Rabinovitch, M. (1991) Toxicol. Pathol. 19, 458-469; Numaguchi, K. (1999) Circ. Res. 85, 5-11; Laufs et al. (2000) Circ. Res. 87, 526-528; Alvarez de Sotomayor et al. (2001) Eur. J. Pharmacol. 415, 217-224; Kuzuya et al. (2004) J. Cardiovasc. Pharmacol. 43, 808-814; Kontaridis et al. (2008) Circulation 117, 1423-1435. Accordingly, it is considered an important target for future therapeutic agents. Budzyn et al. (2006) Trends Pharmacol. Sci. 27, 97-104; Shimokawa et al. (2007) Trends Pharmacol. Sci. 28, 296-302. Activation of RhoA results in downregulation of the myosin light chain phosphatase (MLCP) via upregulation of Rho kinase (ROCK). Halka et al. (2008) Cardiovasc. Pathol. 17, 98-102; Barman et al. (2009) Vasc. Health Risk Manag. 5, 663-671. Inactivation of the MLCP in turn populates its downstream MLC target in its dephosphorylated state, resulting in induction of vasorelaxation.
RhoC has increasingly attracted clinical interest because of the emerging evidence of its metastatic role in inflammatory breast cancer (IBC), which is the most lethal form of locally advanced breast cancer and annually accounts for approximately 6% of the new breast cancer cases in the United States. Jaiyesimi et al. (1992) J. Clin. Oncol. 10, 1014-1024. RhoC has been shown to be overexpressed in ˜90% of human IBC lesions. van Golen et al. (1999) Clin. Cancer. Res. 5, 2511-2519; Clark, E. A., et al. (2000) Nature 406, 532-535. RhoC may also play a role in metastasis of various other tumors including hepatocellular and colon carcinomas. Wang et al. (2003) World J. Gastroenterol. 9, 1950-1953; Bellovin et al. (2006) Oncogene 25, 6959-6967.
Rab GTPases function via their specific effectors (Stenmark et al. (2001) Genome. Biol. 2, reviews 3007.3001-.3007; Jordens et al. (2005) Traffic 6, 1070-1077; Sandilands et al. (2008) Trends Cell Biol. 18, 322-329; Stenmark, H. (2009) Nat. Rev. Mol. Cell Biol. 10, 513-525) as regulators of distinct steps in membrane traffic pathways, including regulation of vesicle formation and movement. As with various Ras GTPases, misregulation of Rab GTPases results in development of a variety of cancers. Stein et al. (2003) Adv. Drug Deliv. Rev. 55, 1421-1437; Chia et al. (2009) Biochim. Biophys. Acta 1795, 110-116.
When small GTPases are redox sensitive, a cellular redox agent functions as their regulator. Heo, J. (2011) Antioxid. Redox Signaling 14, 689-724. Most Rho proteins, including Rac1 and Cdc42, are redox sensitive because they possess a single redox-sensitive cysteine (Cys18, Rac1 numbering) in the GXXXXGK(S/T)C motif (monothiol). Heo, J., et al. (2005) J. Biol. Chem. 280, 31003-31010. A subset of the GXXXXGK(S/T)C motif is found in RhoA and RhoB. Heo et al. (2006) Biochemistry 45, 14481-14489. This subset contains a secondary cysteine (Cys16, RhoA numbering) in addition to the primary redox-sensitive cysteine (Cys20, RhoA numbering, which is equivalent to the Rac1 Cys18) termed the GXXXCGK(S/T)C motif (dithiol). Although both the GXXXXGK(S/T)C and GXXXCGK(S/T)C motifs have the same redox sensitivity (Heo, J. (2005) J. Biol. Chem. 280, 31003-31010) the latter has an additional redox modulation function. Heo et al. (2006) Biochemistry 45, 14481-14489. An analysis of the RhoC crystal structure PDB 2GCO (Dias et al. (2007) Biochemistry 46, 6547-6558) in conjunction with a sequence analysis indicates that RhoC also possesses the GXXXCGK(S/T)C motif. Ras GTPases contain a distinct redox-sensitive NKCD motif. Lander, H. M. (1997) FASEB J. 11, 118-124. Furthermore, the RhoC Cys20 site (the RhoC numbering is the same as the RhoA numbering) is located at the Rho nucleotide-binding site (Dias et al. (2007) Biochemistry 46, 6547-6558), but Ras Cys118 (Harvey Ras numbering) is remote from the Ras nucleotide-binding site. Pai et al. (1989) Nature 341, 209-214. Various Rab family GTPases also possess the GXXXXGK(S/T)C motif (e.g., Rab1, Rab1A, Rab1B, Rab2, Rab2A/B, Rab4, Rab4A/B, Rab8, Rab8A/B, Rab10, Rab13, Rab14, Rab15, Rab19, and Sec4). See Heo, J. (2011) Antioxid. Redox Signal. 14, 689-724.
6-Thiopurine (6-TP) prodrugs, including 6-thioguanine (6-TG), 6-mercaptopurine, and azathioprine, are antimetabolites. They are widely used to treat cancers such as acute lymphoblastic leukemia, acute myeloid leukemia and adenocarcinomas, and autoimmune disorders such as inflammatory bowel disease, Crohn's disease and rheumatoid arthritis, as well as to treat organ transplant recipients. Elion, G. B. (1989) Science 244, 41-47; Langmuir et al. (2001) Best Pract. Res. Clin. Haematol. 14, 77-93; Gearry et al. (2005) J. Gastroenterol. Hepatol. 20, 1149-1157.
In cells, cellular enzymes convert inactive prodrug 6-TPs into the pharmacologically active 6-thioguanine nucleotide that can be grouped into deoxy-6-thioguanosine phosphate (6-TdGNP) and the 6-thioguanosine phosphate (6-TGNP). Elion, G. B. (1989) Science 244, 41-47; Langmuir et al. (2001) Best Pract. Res. Clin. Haematol. 14, 77-93; Gearry et al. (2005) J. Gastroenterol. Hepatol. 20, 1149-1157; McDonald et al. (1996) Cancer Res. 56, 2250-2255; Tiede et al. (2003) J. Clin. Invest. 111, 1133-1145; de Boer et al. (2007) Nat. Clin. Pract. Gastroenterol. Hepatol. 4, 686-694. Furthermore, depending on the number of ribose phosphates, 6-TGNP can be further classified as 6-thioguanosine diphosphate (6-TGDP) and triphosphate (6-TGTP). 6-TdGNP can be incorporated into the de novo synthesis of DNA as a form of 6-TG. 6-TG in DNA can then be recognized as a DNA lesion by the mismatch repair system, which results in induction of the mismatch repair-mediated cell apoptosis. Lage et al. (1999) J. Cancer Res. Clin. Oncol. 125, 156-165; Yan et al. (2003) Clin. Cancer Res. 9, 2327-2334; Karran, P. (2006) Br. Med. Bull. 79-80, 153-170. This 6-TG-mediated induction of mismatch repair is believed to be the main mechanism for the action of 6-TPs in the treatment of acute lymphoblastic leukemia.
In contrast to 6-TdGNP, neither the metabolic path of 6-TGNP nor its therapeutic activity and/or cytotoxicity has been clearly established. A few recent studies have addressed the action of 6-TGNP on small GTPases. It was recently shown that long-term treatment of Ras-activated tumor cells, such as bladder carcinoma (cell-line, T24) and fibrosarcoma (cell-line, HT1080), with 6-TG results in production of cellular 6-TGNP that targets Ras GTPase. Heo et al. (2010) Biochemistry 49, 3965-3976. This Ras-targeting action of 6-TGNP results in downregulation of Ras, which in turn terminates the tumorous growth of these cells. This Ras-targeting action of 6-TGNP extends beyond its effect on tumor cells and is considered cytotoxic because it deregulates the Ras GTP/GDP cycle.
It also has been shown that 6-TGNP targets and inactivates Rho GTPases such as Rac1. Tiede et al. (2003) J. Clin. Invest. 111, 1133-1145; Poppe et al. (2006) J. Immunol. 176, 640-651. This Rho GTPase-targeting action of 6-TGNP may be attributable to the therapeutic effect of 6-TPs on the immune system as well as on inflammatory bowel disease. Cuffari et al. (1996) Can. J. Physiol. Pharmacol. 74, 580-585; Maltzman et al. (2003) J. Clin. Invest. 111, 1122-1124; Quemeneur et al. (2003) J. Immunol. 170, 4986-4995. The therapeutic action of 6-TP in inflammatory bowel disease correlates with the Rac1-targeting 6-TGNP that blocks Rac1 GNE by its GEF Vav (Poppe et al. (2006) J. Immunol. 176, 640-651), although the details of the molecular mechanism by which this occurs are unknown.
Various hormone-related agents and Rho GTPase-downstream effector blockers, as anti-breast tumor drugs, are available. These available drugs are not Rho specific, and thus they also perturb many noncancer-related cellular signaling transductions. However, because these cellular signaling events are vital for cell survival, inhibition/deregulation of these signaling cascades is normally cytotoxic. A desirable chemotherapeutic agent for RhoC-overexpressed IBC is needed that would target RhoC directly and inhibit it. Direct targeting of tumorigenic RhoC by such an agent could reduce cytotoxicity while maximizing the antitumor effect. Similarly, therapeutic agents that directly target and inhibit RhoA and Rac1 activity for treating disorders including blood vessel diseases and autoimmune diseases, respectively, also are needed Inhibitors of various Rab family GTPases are also needed.